The integrins are a group of glycoproteins that are present on a wide variety of cells, where they mediate cell-cell and cell-matrix adhesion via interactions with receptors present on cell membranes or in the extracellular matrix. Known receptors for the various integrin family members include cell surface immunoglobulins, extracellular matrix proteins (laminin, collagen, fibronectin, tenascin), and cadherins.
All known members of the integrin family are composed of two subunits, termed alpha and beta. There are currently at least sixteen recognized alpha subunits and eight different beta-subunits; integrins containing the β1 form of the beta subunit are known as the “β1 integrin family.” Members of this family are expressed by a diverse distribution of tissues and exhibit specific binding specificities. Thus, α1β1 integrin is expressed by T-lymphocytes and fibroblasts and binds to collagen and laminin; in contrast, α4β1 integrin (VLA-4) is expressed by several types of hematopoietic cell and binds to VCAM-1, fibronectin and madCAM. It is therefore the alpha subunit that apparently confers receptor binding specificity to the protein.
A relatively new member of the β1 integrin family, α9β1 (also referred to herein as “alpha-9 integrin”) has been shown to bind to tenascin and osteopontin, both of which are components of the extracellular matrix which are induced at sites of inflammation (Yokosaki; Smith). When sequences of the various alpha subunits were compared, alpha--9 integrin was shown to have the closest sequence identity to the alpha-4 subunit; however this represents only 39% sequence identity (Palmer). Moreover, the two subunits have different cell and tissue distributions. While α9β1 is expressed on airway smooth muscle cells, and non-intestinal epithelial cells (Palmer), and diffusely on hepatocytes and basal keratinocytes (Yokosaki, 1994), α4β1 integrin is present mainly on hematopoietic cells.
Heretofore, there has been no definitive determination of an in vivo function for α9β1 integrin, nor has a physiological consequence of disruption of α9β1-receptor interactions been identified, despite its presence in several tissues, as described above. Nor, despite its association with osteopontin and tenascin, has there been any reason to suspect that alpha-9 integrin might play a role in inflammatory disorders, since the α9β1 molecule had not been associated with any of the hematopoietic cells commonly associated with this disorder.
In studies carried out in support of the present invention, it is now now found that α9β1 is present on neutrophils, a class of phagocytic cells which play an important role in inflammation. In humans, these cells are notable for their relative lack of alpha-4/beta-1 integrin. Therefore, the present invention provides basis for involvement of α9β1 in acute inflammatory responses.
Further differences among the β1-integrins are associated with their binding specificities or endogenous ligands. While they all bind one or more proteins or proteoglycans that form the extracellular matrix, each integrin family member exhibits a distinct molecular specificity which may dictate, in part, its physiological specificity. Thus, while alpha-4/beta-1 integrin is known to bind fibronectin and VCAM-1, alpha-9 integrin has been characterized as binding the matrix proteins osteopontin and tenascin (Yokosaki, 1994; Smith, 1996). According to a further discovery related to the present invention, alpha-9 integrin also binds VCAM-1, though, as discussed below, it is likely that such binding occurs at a site that distinct from the alpha-4 binding site.
The present invention therefore provides basis for new therapeutic regimens directed at modulating alpha-9 integrin binding to its ligand(s), and in particular, those ligands which are involved in the inflammatory response. In addition, it is a further discovery of the present invention that many of the compounds or drugs that modulate (inhibit or enhance) alpha-4/beta-1 integrin binding also modulate alpha-9 integrin binding. This discovery therefore provides new pharmaceutical compositions and methods of treatment for modulating alpha-9 integrin binding, as well as screening methods for identifying new alpha-9 integrin modulatory compounds.